Repression of untimely blood coagulation is the therapeutic option of choice for acute treatment and long-term prevention of thrombolic events. Among the anti-coagulants coumarins are widely used for the prevention of thrombosis such as in patients immobilized after surgery, patients having a chronic heart failure, patients having atherosclerotic vascular disease, patients having a malignancy, and patients that are pregnant. Moreover, coumarins are the most widely used oral anticoagulants for the treatment and prophylaxis of thrombosis [Suttie, 1987]. Coumarins are typically derivatives of 6-hydroxycoumarin, such as 3-(acetonylbenzyl)-4-hydroxycoumarin (COUMADIN®).
The coumarins target the blood coagulation cascade indirectly by inhibition of the vitamin K cycle.
Vitamin K is an essential cofactor for the post-translational activation by gamma-carboxylation of a group of regulatory proteins, the Gla-proteins. In several metabolic pathways, some key proteins require carboxylation for proper function. The blood coagulation cascade is the best-studied example. Here, the procoagulant factors II, VII, IX and X, and the anticoagulant factors protein C, protein S, protein Z are dependent on gamma-carboxylation. This post-translational modification enables the attachment of the modified proteins—in the presence of calcium—to phospholipid-bilayer membranes which is an essential step in the activation of blood coagulation [Sperling et al., 1978] [Esmon et al., 1975]. Other proteins requiring gamma-carboxylation are the matrix gla protein and osteocalcin, both regulators of bone metabolism [Price, 1988] and the “growth arrest specific gene”, a signal transduction protein of the cell cycle [Manfioletti et al., 1993] [Stitt et al., 1995].
During gamma-carboxylation, a carboxyl group is introduced into glutamate residues of the target proteins by the enzyme gamma-glutamyl carboxylase (GGCX) in liver microsomes [Furie & Furie, 1988] [Suttie, 1987]. The reaction requires as a cofactor stoichiometric amounts of reduced vitamin. K1 hydroquinone (vitamin K1H2) which is oxidized to vitamin K-2,3 epoxide [Cain et al., 1997]. The regeneration of the active cofactor is mediated by a multi-protein complex termed vitamin K-2,3-epoxide reductase (VKOR) [Wallin & Martin, 1985]. The same complex is targeted by the coumarin-type poisons used in rodent pest control. This “vitamin K cycle” has been characterized biochemically in great detail but the molecular components have not yet been purified to homogeneity [Guenthner et al., 19981]. Moreover, the molecular nature of coumarin activity and the molecules interacting with coumarins are still elusive.
It is generally appreciated in the art that although largely effective, there are a number of limitations to the use of coumarins. First of all; there are humans that are inert to coumarin treatment. The term warfarin resistance (WR) is used for individuals who maintain normal clotting factor activities despite oral anticoagulation by coumarins (OMIM Access. No. 122700). Autosomal dominant transmission has been observed in several pedigrees [O'Reilly et al., 1964] [O'Reilly, 1970]. Combined deficiency of all vitamin K dependent clotting factors (VKCFD) is a very rare bleeding disorder in humans of autosomal recessive inheritance with 14 cases described as yet [McMillan & Roberts, 1966] [Fischer, 1966] [Johnson et al., 1980] [Goldsmith et al., 1982] [Vicente et al., 1984] [Ekelund et al., 1986] [Pauli et al., 1987] [Leonard, 1988] [Pechlaner et al., 1992] [Boneh & Bar-Ziv, 1996] [Brenner et al., 1998] [Spronk et al., 2000] [Oldenburg et al., 2000]. Clinical symptoms of the disease include episodes of perinatal intracerebral hemorrhage sometimes with fatal outcome. The bleeding tendency is usually completely reversed by oral administration of vitamin K. Additional symptoms in newborns can resemble warfarin embryopathy with nasal and distal phalangeal hypoplasia and premature calcification of epiphyses [Pauli et al., 1987]. The disease may result either from a defective resorption/transport of vitamin K to the liver [Prentice, 1985] or from mutations in one of the genes involved in gamma-carboxylation. In subtype 1 (VKCFD1, OMIM # 277450), mutations in the GGCX gene on chromosome 2p12 result in insufficient carboxylation of clotting factors [Brenner et al., 1998] [Spronk et al., 2000]. There has been described a linkage of two kindreds with familial multiple coagulation factor deficiency (FMFD, now re-named: VKCFD2, OMIM #607473) to a 20 Mb interval of the pericentric region of chromosome 16p12-q21 [Fregin et al., 2002]. Patients with VKCFD2 showed significantly increased serum levels of vitamin K epoxide, thus suggesting a defect in one of the subunits of the VKOR complex. Taken together, there is evidence that there are patients that display warfarin resistance. As a result, there is a need to identify novel coumarins derivatives that are effective anticoagulants for treating these patients, and methods for identifying these coumarin derivatives.
The use of coumarins is associated with a risk of spontaneous bleedings, with a significant mortality rate. Moreover, the prediction of the accurate coumarin maintenance dose is difficult. In the absence of the target molecule which coumarin exerts an effect on, the treatment regimen has to be established, on a patient-by-patient basis. During the time the optimum regimen is yet not established the patient either suffers from an increased risk of thrombogenesis or of an increased risk of bleeding. Therefore there is a need for a method of determining the optimal treatment regimen that is faster and saver. Further, establishing an optimal treatment regimen is complicated by the fact, that there is a considerable delay between the administration of coumarins and the onset of its anticoagulant activity. Given the delayed action of coumarin and given the fact that coumarin tends to accumulate in time there is a need for coumarin derivatives that effect blood coagulation faster than the coumarins known in the art. By the same token there is also a need for coumarins that are metabolized more rapidly so that accumulation of coumarin may be prevented or ameliorated and as a result the danger of overdosing is decreased or abolished.
It is well appreciated that if coumarin treatment is initiated during a thrombic state, the levels of protein C and S decline, thus temporarily creating a thrombogenic potential which is usually compensated for by overlapping heparin and coumarin administration for a number of days. Again, there is a need to identify the molecular target of coumarin action in order to be able to screen for novel coumarin derivatives that do not possess these limitations or at least to a lesser extend.
A coumarin therapy sometimes induces skin necrosis in patients and if applied during pregnancy may cause embryopathy creating a need for novel coumarin derivatives which do not cause these effects.
There are a number of interactions between drugs and coumarins. Some of these drugs like Phenobarbital induce lower plasma levels of coumarins due to an increased metabolization of coumarin which is believed to be caused by the mixed-function oxidases like the cytochrome P450 mixed-function oxidases. Such interaction is of clinical relevance if the appropriate regimen of e.g. Phenobarbital and coumarin has been determined and later on only administration of Phenobarbital is discontinued leading to a rise of the plasma level of coumarin which causing excessive anticoagulation. Other drugs like Amiodarone cause a delayed metabolization of coumarin leading again to excessive anticoagulation if co-administered with coumarins. Since the molecules affected by coumarins are not known in the art there is a need to develop novel coumarins and tools to identify the latter in order to solve these problems.
Finally, coumarins, especially warfarin, are not only used in humans but since the 1950s, coumarins have been in use as an active ingredient in rodenticidal compositions. The basis for the effectiveness of warfarin as a rodenticide lies in the fact that it is an effective anti-coagulant in small, multiple doses. One or two doses of the compound are seldom fatal if taken at the recommended concentration; thus the hazard of acute toxicity to man, domestic animals, and wildlife is greatly reduced. Usually the rodents begin to die after four or five daily doses of the materials, and the population is greatly reduced or eradicated in approximately three weeks. Death is caused by hemorrhages, brought about by the action of the warfarin in reducing the clotting power of the blood. These hemorrhages may be external or internal and can be initiated by very slight injury or capillary damage. One of the other advantages of coumarins is that, because multiple ingestions are required to kill the rodents, they do not develop bait shyness. Beginning in 1969, rodents—particularly rats and, to a somewhat lesser extent, mice—began showing resistance to warfarin baits. The general assumption was that such resistance had a genetic basis. As for the mechanism, it is the VKORC1 complex mentioned above that is targeted by derivatives of warfarin in use for rodent pest control [Jackson et al., 1988]. Resistance to coumarin derivatives has arisen spontaneously in several wild rodent populations rendering the use of these drugs locally ineffective for pest control. Autosomal dominant loci for warfarin resistance have been mapped in the mouse (War) to chromosome 7 [Wallace et al., 1976] and in rat (Rw) to the long arm of chromosome 1[Greavses & Ayres, 1967] [Kohn & Pelz, 1999]. Since the VKOR complex is the target of the coumarin drugs resistance is thought to be mediated by alterations in one of its protein components [Jackson, 1988]. The development of resistance in rodents has created a need for identifying the target of coumarins action which would facilitate the development of novel coumarin-derivatives for use in pest control.
Taken together it is an object of the present invention to provide a target molecule for coumarin and its derivatives in mammals. It is another object of the present invention to provide methods for identifying novel coumarins which solve at least one of the problems mentioned above. It is a further object of the present invention to identify polypeptides and nucleic acids coding for them which cause warfarin resistance in human and non-human mammals, preferably rodents. It is also an object of the present invention to diagnose, prevent and/or treat disorders and diseases selected from diseases from warfarin resistance, familial multiple factor deficiency, a disorder or disease associated with increased blood coagulation such as patients suffering from a thrombus and/or patients having an increased risk of developing a thrombus, such as an inherited increased risk of thrombogenesis, preferably an increased risk of thrombogenesis due to a surgery or due to pregnancy, and increased vascular calcification. Moreover, it is also an object of the present invention to diagnose, prevent and/or treat diseases or disorders associated with attenuated blood coagulation, such as hemophilia, disorder associated decreased vascular calcification and disorders and diseases with an increased risk of bleeding. Finally, it is an object of the present invention to provide a method for identifying coumarin and its derivatives which are effective in pest control of non-human mammal and compositions for killing rodents.